Project Summary Acute or chronic stress contributes to both escalation of alcohol use and relapse to alcohol seeking after abstinence. Stressors are hypothesized to contribute to relapse in part by promoting responses to cues in the environment that have been previously associated with alcohol availability, yet the brain mechanisms of this interaction between cues and stress are poorly understood. An area of the brain implicated in both stress- and cue-induced reward seeking is the ventral pallidum (VP). I have previously found that VP neurons encode the vigor of cue-elicited reward seeking, and my preliminary results suggest that VP neurons encode reinstatement of reward seeking by the pharmacological stressor, yohimbine. In the current proposal, I aim to confirm and expand my findings regarding VP encoding of reinstatement by stress, using a behavioral stressor, and to further dissect the neural circuit mechanisms by which VP neurons contribute to alcohol seeking. Here, in the K99 phase, I will examine VP encoding of cued alcohol seeking and reinstatement induced by stress in dependent versus non-dependent alcohol seeking rats. This aim will provide me with new training in models of alcohol dependence and stress reinstatement. I will then examine encoding of alcohol seeking behavior in inputs to VP from the prefrontal cortex (PFC) and basolateral amygdala (BLA), using a novel method for projection specific measurement of activity. This method, fiber photometry, uses viral-based expression of a fluorescent calcium indicator paired with optical measurement of fluorescence. Training in this skill will allow me to measure projection-specific neural activity in the proposed aims and future projects. Further, this aim will provide critical information regarding the timing of activity in BLA and PFC inputs during alcohol seeking, to be tested in the R00 phase. I will also assess the collateralization of these neurons, comparing two methods: traditional sectioning and immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). This method provides unprecedented access to the three-dimensional structure of immunolabeled cells, and the opportunity to obtain both large-scale and high resolution information simultaneously, crucial for planned experiments in my future laboratory. In the R00 phase I will test the functional role of temporally precise activity in BLA and PFC inputs to VP during cued alcohol seeking and reinstatement of alcohol seeking, using optogenetics. I will also measure activity in genetically-defined populations of neurons projecting from the nucleus accumbens (NAc), which include both D1- and D2-dopamine receptor dominant neurons. Using transgenic rats I will selectively express calcium indicators in either D1- or D2-dominant neurons projecting to VP. These experiments will provide novel data regarding the differential activity of D1 and D2 neurons projecting to VP and will provide an important framework for future experiments and grants. These experiments will characterize the neural circuit mechanisms by which VP neurons drive alcohol seeking, and how they differ across different states of dependence and following stress.